Pollen production is essential to the sexual reproductive success of the flowering plant. Male gametogenesis is a highly regulated developmental process that occurs within the diploid sporophytic tissue of the anther. It comprises three major phases: the differentiation of the sporogenous cells and meiosis; the development of the free uninucleate microspores; and the pollen maturation following microspore mitosis and ending with the formation of mature pollen (Scott, R., Hodge, R., Paul, W., Draper, J. Plant Sci. 80:167-191 (1991)). Typically, pollen captured by a receptive stigma of the pistil will germinate and the pollen tube will grow extracellularly through the stigma and style until it reaches the ovule where it releases its nuclei that effect double fertilization. Similarly to a seed, the pollen accumulates reserves that enable it to germinate on a receptive stigma.
Normal pollen development is dependent upon the tapetum, a cellular layer lining the locular space of the anther. The tapetum provides the developing microspores with nutrients and other necessary products such as enzymes and structural components (Pacini, E., Franchi, G. G., Hesse, M. Plant Syst. Evol. 149:155-185 (1985)). In Brassica, the secretory tapetum is made up of cells which are metabolically very active until about microspore mitosis at which time they degenerate (Grant, I., Beversdorf, W. D., Peterson, R. L. Can. J. Bot. 64:1055-1068 (1986); Murgia, M., Charzynska, M., Rougier, M., Cresti, M. Sex. Plant Reprod. 4:28-35 (1991); Polowick, P. L., Sawhney, V. K. Sex. Plant Reprod. 3:263-276 (1990)). When the tapetal cells degenerate they release their cellular contents into the anther locule where they are thought to contribute to the formation of the external pollen coat (Evans, D. E., Taylor, P. E., Singh, M. B., Knox, R. B. Planta 186:343-354 (1992); Heslop-Harrison, J. New Pytol. 67:779-786 (1968)). The pollen coat (sporoderm) consists of two layers, the exine (outer wall) and the intine (inner wall). The exine can be further subdivided into the nexine and sexine layers and is often elaborately sculptured and patterned (Scott, R. J. In: Molecular and Cellular Aspects of Plant Reproduction (eds) Scott, R. J., Stead, M. A. 55:49-81 (1994)).
The interstices of the exine contain various substances including proteins, enzymes, lipids and allergens (Knox, R. B. In: Embryology of Angiosperms, (ed) Johri, B. M. pp. 197-271 (1994)) many of which are of tapetal origin. The lipidic and proteinaceous layer coating the exine is also called the tryphine. The mature pollen grain released upon anther dehiscence is dry and the drying process causes the tryphine to retract into the exine cavities. Numerous pollen enzymes have been identified (Brewbaker, J. L. In: Pollen: Development and Physiology (ed) Heslop-Harrison, J. pp. 156-170 (1971); Hiscock, S. J., Dewey, F. M., Coleman, J. O. D., Dickinson, H. G. Planta 193:377-383 (1994); Knox, R. B. In: Pollen: Development and Physiology (ed) Heslop-Harrison, J. pp. 171-173 (1971); Lavithis, M., Bhalla, P. L. Sex. Plant Reprod. 8:289-298 (1995); Travis, J., Whitworth, T., Matheson, N., Bagarozzi, D. Acta Biochim. Pol. 43:411-418 (1996)). Many of these enzymes are located in the pollen coat especially in the intine layer and are readily elutable from the pollen grain. Some of these enzymes such as pectate lyases and ribonucleases have been shown to correspond to pollen allergens (Knox, R. B., Suphioglu, C. Sex. Plant Reprod. 9:318-323 (1996)).
Recently, genes encoding some of the pollen coat proteins have been isolated. The PCP7 gene encodes a pollen coat peptide from Brassica oleracea that has been shown to interact with S-locus glycoproteins (Doughty, J., Hedderson, F., McCubbin, A., Dickinson, H. Proc. Natl. Acad. Sci. USA 90:467-471 (1993); Hiscock, S. J., Doughty, J., Willis, A. C., Dickinson, H. G. Planta 194:367-374 (1995)). The PCP1 gene encodes a cysteine-rich protein which may be involved in pollen-stigma interactions in Brassica oleracea and which belongs to a family of 30 to 40 genes (Stanchev, B. S., Doughty, J., Scutt, C. P., Dickinson, H., Croy, R. R. D. Plant J. 10:303-313 (1996)). This gene was shown to be expressed gametophytically and its product is released from the pollen protoplast into the surface coating.
There have also been numerous genes isolated which show expression in the tapetum, yet the function of the proteins they encode (Schrauwen, J. A. M. Acta Bot. Neerl. 45:1-15 (1996)), and whether they associate with the pollen coat is largely unknown. However, genes encoding β-1,3-glucanase have been shown to be expressed in the tapetum and these enzymes are involved in breaking down the callose wall surrounding the tetrads releasing the microspores (Bucciaglia, P. A., Smith, A. G. Plant Mol. Biol. 24:903-914 (1994); Hird, D. L., Worrall, D., Hodge, R., Smartt, S., Paul, W., Scott, R. Plant J. 4:1023-1033 (1993)). There are also some examples of tapetal-specific genes (ie. expressed sporophytically) whose products were shown to be localized to the pollen coat. The related genes Satap35 and Satap44 from Sinapis alba are associated with the exine of the developing microspore and may be involved in sporopollenin formation and/or deposition (Staiger, D., Kappeler, S., Müller, M., Apel, K. Planta 192:221-231 (1994)). Recently the pollen coat localized male determinant of self-incompatibility has been identified in B. campestris (B. rapa) and B. oleracea as the S-locus cysteine rich protein SCR/SP11 which is expressed in the tapetum (Schopfer, C. R., Nasrallah, M. E., Nasrallah, J. B. Science 296:1697-1700 (1999); Takayama, S., Shiba, H., Iwano, M., Asano, K., Hara, M., Che, F. -S., Watanabe, M., Hinata, K., Isogai, A. Proc. Natl Acad. Sci. USA 97: 1920-1925 (2000); Kachroo, A., Schopfer, C. R., Nasrallah, M. E., Nasrallah, J. B. Science 293: 1824-1826 (2001); Shiba, H., Takayama, S., Iwano, M., Shimosato, H., Funato, M., Nakagawa, T., Che, F. -S., Suzuki, G., Watanabe, M., Hinata, K., Isogai, A. Plant Physiol. 125: 2095-2103 (2001); Shiba, H., Iwano, M., Entani, T., Ishimoto, K., Shimosato, H., Che, F. -S., Satta, Y., Ito, A., Takada, Y., Watanabe, M., Isogai, A., Takayama, S. Plant Cell 14: 491-504 (2002)). Similarly, the predominant protein on the surface of maize pollen is an endoxylanase synthesized by the tapetum (Bih, F. Y., Wu, S. S. H., Ratnayake, C., Walling, L. L., Nothnagel, E. A., Huang, A. H. C. J. Biol. Chem. 274: 22884-22894 (1999)).
The Sta 41-2 and Sta 41-9 genes from Brassica napus encode proteins that possess a hydrophobic domain similar to that of the seed oleosins (Robert, L. S., Gerster, J. L., Allard, S., Cass, L., Simmonds, J. Plant J. 6:927-933 (1994a)). Sequence similarity among the Sta 41-2 and Sta 41-9 genes, and seed oleosin genes from Brassica napus (Murphy, D. J., Prog. Lipid Res. 32:247-280 (1993)) are limited to the relatively small hydrophobic domain and show levels of 30-36% identity. These tapetally expressed genes have now been demonstrated to belong a large family of related anther oleosin-like genes in Brassica (Ross, J. H. E., Murphy, D. J. Plant J. 9:625-637 (1996); Ruiter, R. K., Van Eldik, G. J., Van Herpen, R. M. A., Schrauwen J. A. M., Wullems, G. J. Plant Cell 9:1621-1631 (1997)). Unlike the other tapetally expressed pollen coat localized proteins mentioned above, the oleosin-like proteins do not possess a signal peptide and are thought to be released passively into the anther locule upon tapetum degeneration by association with lipids released from the tapetum or found as part of the tryphine of the pollen coat. Without wishing to be bound by theory, the hydrophobic region of the tapetal oleosin-like protein may be required for localization upon the pollen coat by association with lipids. The tapetal oleosin-like proteins constitute the major protein of the Brassica pollen tryphine and they occur as post-translationally cleaved protein products (Ross, J. H. E., Murphy, D. J. Plant J. 9:625-637 (1996)). The function of the tapetal oleosin-like proteins is unknown but they may play a role in the interaction between the pollen and the stigma the specialized part of the pistil that receives the pollen.
The stigma is responsible for capturing and selecting compatible pollen grains and for facilitating their germination. Angiosperm stigmas have been classified morphologically as ‘dry’ stigmas having an extracuticular proteinaceous pellicle but no free-flowing secretion or ‘wet’ stigmas which are covered by a secretion at the receptive stage (Heslop-Harrison, Y., Shivanna, K. R. Ann. Bot. 41:1233-1258 (1977)). In Brassica, the dry stigma is the site of the sporophytic self-incompatibility (SI) response with incompatible pollen being unable to grow through the stigmatic papillar cells or failing to germinate altogether.
A number of genes have been shown to be preferentially expressed in the Brassica stigma and most of these genes correspond to genes associated with SI: SLG (S-locus glycoprotein), SRK (S receptor kinase; U.S. Pat. No. 5,484,905) or SLR (S-locus-related; WO94/25613) genes (for review: Nasrallah, J. B., Nasrallah, M. E. Plant Cell 5:1325-1335 (1993)). The products of the SLG and SRK genes are believed to be involved in a signal pathway modulating the SI reaction in response to a ligand carried by the pollen grain. WO94/25613 is directed to pistil-, and anther-specific gene expression. It discloses the cloning of several SLG's genes and the isolation of the SLG1 promoter region, and the preparation of transcriptional fusion products using the promoters from the SLG genes. Furthermore, U.S. Pat. No. 5,585,543 discloses several genes related to the S-locus.
Another example of a gene highly expressed in the Brassica stigma is Pis 63 (Robert, L. S., Allard, S., Gerster, J. L., Cass, L., Simmonds, J. Plant Mol. Biol. 26:1217-1222 (1994b)). The promoter obtained from the genomic clone PISG 363, which contains gene Pis 63-2 was shown to direct the expression of the marker gene β-glucuronidase transiently in B. napus stigmas and stably in the stigmas of transformed tobacco plants (Robert, L. S., Lévesque-Lemay, M., Gerster, J. L., Hong, H. P., Keller, W. Plant Cell Rep. 18:357-362 (1999)).
The SI response in Brassica provides an example that a molecular based interaction between the pollen grain and the stigmatic papillae exists and that such an interaction can be modified or mimicked by targeting polypeptides to the appropriate part of the pollen and/or stigma. It is thought that localization of the SLG proteins arrises as a result of the appropriate signal peptide directing the protein extracellularly, following expression.
The preparation of plants with female sterility based on a style-stigma specific “STMG” gene and derived constructs using PSTMG promoter cassettes is disclosed in U.S. Pat. No. 5,633,441. These constructs include transcriptional fusions comprising barnase, papain or RNAse. In U.S. Pat. No. 5,652,354, the use of stamen-selective promoters useful in driving expression in anther, pollen, or filament cells, especially in the tapetum or anther epidermal cells is disclosed. U.S. Pat. No. 5,571,904 is directed to male flower specific gene sequences. Genomic clones of pMS10, 14 and 18 were obtained and promoter cassettes were constructed using MS10. There is also evidence presented where pMS14 expression has been localized within the tapetal cell layer. Other publications also disclose floral-specific genes and associated regulatory elements. For example, U.S. Pat. No. 5,633,438 discloses microspore-specific regulatory element, Bnm1; U.S. Pat. No. 5,545,546 discloses the cloning of W2247, a pollen specific promoter obtained from maize (inbred maize line W22); U.S. Pat. No. 5,659,124 teaches use of existing anther specific promoters to produce male sterile plants; WO92/13957 is directed to the cloning of CA444 which is a stamen/anther specific gene; WO97/13401 discloses the cloning of a rice tapetal specific gene RTS2; WO93/25695, is directed to the preparation of male sterile plants using tapetal specific promoters such as those from the TA29 gene or PT72; CA 2,099,482, teaches the disruption of the formation of viable pollen resulting in male sterile plants using an anther specific promoter; CA 2,106,718 is directed to the disruption of normal pollen development using anther specific promoters driving chimeric constructs that disrupt pollen development; Worrall, D., Hird, D. L., Hodge, R., Paul, W., Draper, J., Scott, R. Plant Cell 4:759-771 (1992)) teaches the use of a tapetal specific promoter to drive the expression of callase which prematurely degrades the callose wall surrounding the developing tetrad of microspores thereby releasing the microspores into the anther locule. This premature release of microspores leads to male sterility. CA 2,165,934 discloses the use of a polygalacturonase promoter to drive a chimeric construct within microspores of Brassica napus plants. However, there is no teaching of modifying the extracellular domain of a free microspore or pollen grain.
Based upon the review of the prior art, there are two known mechanisms that exist for the targeting of a protein onto the pollen coat, either by deposition following tapetal degradation, or as a result of extracellular targeting either from the tapetal cells or microspore cells via a signal peptide. Similarly, extracellular targeting of pistil-derived gene products, for example the SLG gene product, appears to involve the use of a signal peptide. However, none of the prior art publications disclose modifying the protein composition of the microspore/pollen coat or the interactions of these proteins with the stigma or pistil. Rather, most of the published literature is directed to producing sterile plants through the disruption of pollen development, although this disruption does not occur by modifying the extracellular compartment. Nor does the prior art teach a similar modification of the stigma cells using chimeric gene constructs that would affect the interaction between these cells and a modified pollen grain, while all of these cell types could remain viable. The approach described herein is primarily directed at modifying pollen or stigma function, and in some instances affects the interaction between pollen and stigma.
There is no teaching of the preparation of transcriptional or translational fusion proteins specifically designed to localize on the exterior of a pollen or stigma cell. For example, but not limited to, comprising hydrophobic domains of pollen coat proteins and the like, to direct the translocation of the fusion product to the exterior surface of the pollen. Furthermore, beyond uses that are directed to pollen disruption for the production of sterile plants, the prior art does not disclose methods that provide for peptide display, antibody production, altering the food value of pollen for human consumption, the use of treating insects, or alleviating allergenic responses by specifically targeting protein products to the surface of the appropriate floral cell.
This invention relates to a method of modifying the extracellular compartment of floral cells, including targeting proteins, protein fusions, fragments thereof, or peptides to this extracellular domain. Methods using chimeric gene constructs that allow targeting of proteins, fusion proteins or peptides of interest to cells of the pistil, microspore, or to the pollen coat to modify floral functions or interactions are disclosed and exemplified.